Purification and mass spectrometric analysis of the μ opioid receptor

A mouse μ opioid receptor was engineered to contain a FLAG epitope at the amino-terminus and a hexahistidine tag at the carboxyl-terminus to facilitate purification. Selection of transfected human embryonic kidney (HEK) 293 cells yielded a cell line that expressed the receptor with a B _max of 10 pmol/mg protein. ³[H]Bremazocine exhibited high affinity binding to the epitope-tagged μ opioid receptor with a K _D of 1.0 nM. The agonists [D-Ala²,N-Me-Phe ⁴,Gly⁵-ol]enkephalin (DAMGO), morphine and [D-Ala ²,D-Leu⁵]enkephalin (DADL) competitively inhibited bremazocine binding to the tagged μ receptor with K_I's of 3.5, 17 and 70 nM, respectively. Chronic treatment of cells expressing the epitope-tagged μ receptor with DAMGO resulted in down-regulation of the receptor, indicating that the tagged receptor retained the capacity to mediate signal transduction. The μ receptor was solubilized from HEK 293 cell membranes with n-dodecyl-β-D-maltoside in an active form that maintained high affinity bremazocine binding. Sequential use of wheat germ agglutinin (WGA)-agarose chromatography, Sephacryl S300 gel filtration chromatography, immobilized metal affinity chromatography, immunoaffinity chromatography, and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) permitted purification of the receptor. The purified μ opioid receptor was a glycoprotein that migrated on SDS/PAGE with an apparent molecular mass of 80 kDa. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was used to identify and characterize peptides derived from the μ opioid receptor following in-gel digestion with trypsin or chymotrypsin, and precursor-derived tandem mass spectrometry (ms/ms) confirmed the identity of several peptides derived from enzymatic digestion of the μ opioid receptor.